The goal of this application is to elucidate properties of myelodysplastic syndrome (MDS) stem cells that will lead to improved strategies for therapeutic targeting. In the context of MDS, malignant stem cells are thought to reside at the heart of disease, driving progression and ultimately transformation to acute myeloid leukemia (AML). While some drugs have activity in MDS, few if any clinically approved agents are known to efficiency eradicate MDS stem cells. Thus, our studies have focused on fundamental aspects of cellular metabolism as a potential entry point for developing novel therapies. It has become increasingly clear that normal hematopoietic stem cells (HSCs) display unique metabolic properties that distinguish them from other cells in the hematopoietic system. We propose that MDS stem cells are similarly unique and employ metabolic mechanisms that are distinct from bulk MDS cells as well as normal stem/progenitor cells. Indeed, our preliminary data show that like normal HSCs, most MDS stem cells are quiescent and reside in a state of relatively low reactive oxygen species (termed ?ROS-low?). Intriguingly though, during pathogenesis MDS stem cells shift to an energy metabolism phenotype characterized by preferential reliance on oxidative phosphorylation. During the same transitional phase, MDS stem cells also strongly up-regulated protein synthesis pathways. These distinct biological properties render MDS stem cells susceptible to therapeutic agents such as venetoclax and omacetaxine. Indeed, our preclinical modeling studies have been so successful with these agents that two new clinical trials have been developed to test their use for high-risk MDS. Both trials recently opened and are now accruing. For the current proposal we intend to perform a detailed analysis of MDS stem cells in the patients treated from these trials. We will also conduct further preclinical modeling to better characterize the mechanism by which MDS stem cells can be selectively eradicated. In addition to high-risk stages of disease, our studies will be extended to include patients with low and intermediate risk MDS. Taken together, the proposed research will elucidate the key metabolic pathways that mediate survival of MDS stem cells and identify novel strategies for treatment of MDS patients.